Journal: Scientific Reports

Article Title: Angiotensin II induces kidney inflammatory injury and fibrosis through binding to myeloid differentiation protein-2 (MD2)

doi: 10.1038/srep44911

Figure Lengend Snippet: Effects of MD2 blockade by ( A) L6H21 or ( B) siRNA-mediated knock-down on Ang II-induced MD2/TLR4 complex formation were determined. NRK-52E cells were pretreated with L6H21 (2.5, 5, 10 μM) for 1 h, or transfected with 1 μg MD2-specific siRNA (Methods), stimulated with Ang II (1 mM) for 30 min, and cell lysates were co-immunoprecipitated (IP) with anti-MD2 or -TLR4 antibodies, and Western blot analysis (IB) made to detect TLR4 or MyD88; n = 4 independent determinations. The gels were run under the same experimental conditions. Shown are cropped gels/blots (The gels/blots with indicated cropping lines are shown in ). ( C ) The effects of L6H21 on Ang II binding to cell surface was determined. NRK-52E cells were incubated with Ang II conjugated with fluorescent FITC (FITC-Ang II; 50 mg/ml) with or without L6H21 (2.5, 5.0, 10 μM), and flow cytometry was used to detect cell-bound fluorescence; shown is a representative flow analysis with the median fluorescence intensity (MFI) indicated for each group; n = 4 independent determinations.

Article Snippet: Ang II labelled with FITC was purchased from Bankpeptide Biological Technology Co. (Hefei, Anhui province, China).

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Incubation, Flow Cytometry, Fluorescence